Gene carrier penetration through the CF sputum barrier in the airways is a prerequisite for successful gene therapy with aerosolized gene carriers for CF. However, we found that the leading viral and nonviral gene carriers for CF that we have tested to date are incapable of penetrating the highly viscous and elastic sputum obtained from the airways of CF patients. This finding may help explain the disappointing clinical results with gene therapy in the airways, and points to a dire need to discover new approaches that facilitate viral and non-viral gene carrier penetration through CF sputum. Our overall hypothesis is that improved sputum penetration by viral and non-viral gene carriers may be achieved by: (i) modulating the biophysical properties of CF sputum, and/or (ii) masking the adhesive portions of gene carriers or of CF sputum itself with simple chemicals with a history of safe use in humans. This proposal will utilize powerful biophysical techniques to first quantify the transport of leading viral and nonviral gene carriers through undiluted and unmanipulated sputum expectorated by CF patients, including AAV (serotypes 1,2,5) from Project 1 and Core A, recombinant AAV vectors provided by Project IV, compacted DNA nanoparticles currently in human clinical trials, and non-mucoadhesive variations of compacted DNA nanoparticles engineered by our lab (Aim 1 A). We will also investigate the mechanisms responsible for restricting rapid penetration of gene carriers by investigating the microstructure and nano-scale rheological behavior of CF sputum as probed by the various particles from Aim 1A (Aim1 B). Subsequently, we will test potentially synergistic methods by which to decrease the barrier properties of sputum that hinder gene carrier transport, including: (i) combinations of mucolytic agents that target major components of sputum (Aim 2A),(ii) pre-administration of sacrificial particles that create large openings in CF sputum (Aim 2B),and (iii) a chemical coating that minimizes adhesion of gene carriers to sputum (Aim 2C). If successful, our work will provide fundamental insights into the CF sputum barrier, identify new strategies that allow promising gene carriers to overcome theformidable CF sputum barrier, and lead to strongly improved gene delivery efficiency RELEVANCE (Seeinstructions): Penetration of gene carriers through thick mucus secretions is a requirement for successful gene therapy for Cystic Fibrosis (CF). We have discovered that several gene delivery systems currently being considered for the potential cure of CF are incapable of penetrating these mucus secretions. We seek to test novel strategies to improve the penetration rates of promising viral and nonviral gene carriers for CF gene therapy.